Method development

N-glycosylation analysis of glycoproteins can be performed in different ways by LC-MS: the intact proteins, proteolytic digests or released N-glycans can be analyzed. Released glycans can be derivatized to even the ionization efficiency and thereby to improve the accuracy of quantification. This approach provides excellent coverage of the N-glycosylation pattern. We reported previously a method to identify and quantify 2-AA labeled N-glycans by ion-trap MS after RPC.(17) The method is selective for many N-glycan isomers and its robustness and reproducibility have been demonstrated. However, the method was developed to analyze N- glycans of mAbs in advanced development stages, not for early development when limited sample amounts are available and higher sensitivity is required. Thus, we have started to develop a nanoLC-MS method.

Our approach used glycans released from RNase B (Figure S1 and Table S1), a model protein for N-glycosylation analysis. RNase B N-glycans were prepared as described in the methods section, and the labeled and purified N-glycans were analyzed by nanoLC-MS. The nanoLC was configured in “direct injection on a nano column” mode because highly hydrophilic 2-AA N-glycans like the high mannose type glycans did not bind properly to the trapping column, and were therefore underrepresented in setups that included a pre- concentration step with a trapping column. The user defined injection routine allows injection volumes from 1 to 4 µl without major gradient delay. This is achieved by, depending on the chosen injection volume, switching the sample loop between 5 to 15 min into the flow to ensure that the entire sample leaves the loop before switching it back to loading position. Because of the direct injection, samples must be highly purified to avoid salt plugs entering the nanospray chamber of the MS, which may damage the emitter tip and shorten its lifetime. The chosen acidic mobile phases resulted in high selectivity for many glycan isomers on RP and improved ionization of glycans in the positive ionization mode. The portion of formic acid in the mobile phase could be lowered to 0.5% compared with 1% for the LC-MS method, which may be due to the more efficient ionization in the nano spray. The 2-AA glycans occurred mostly as double [M+2H]2+ charged ions, 2-AA N-glycans smaller than 1500 Da occurred as single [M+H]1+ charged ions. In addition along with protonated ions, mixed adduct ions with sodium (e.g., [M+H+Na]2+) or potassium (e.g., [M+H+K]2+) were also present. The 2-AA glycans were identified by MS, MS² and MS³. In addition to RNase B, the method was tested with multiply branched and sialylated glycans to cover all types of glycans. Therefore, several N-glycan standards were labeled with 2-AA and analyzed (Figure S2). All types of N-glycans can be identified and quantified with this new approach, which is in agreement with our previously reported RP LC-MS approach. (17) High mannose glycans

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elute first, followed by non-fucosylated hybrid and complex variants, then fucosylated hybrid and complex glycans. The overall chromatographic resolution is higher for the nanoLC approach compared with the LC-MS approach.

As described in the introduction, the amount of sample available can be very limited at various stages of biopharmaceutical development, especially during early development phases like pool or clone selection. During clone selection, numerous samples in complex matrices (cell culture supernatant) must be analyzed, making affinity purification necessary. Therefore, sample preparation had to be adapted to increase throughput and include affinity purification steps. 96-well plate-based sample preparation is a viable option, the success of which has already been demonstrated for glycoprotein analytics.(10, 15, 30) We selected a centrifugation-based 96-well filter plate sample preparation because we had observed inhomogeneous flow through the small scale columns in the filter plate wells on a vacuum manifold. Protein A Sepharose, which is commercially available and state-of-the-art for downstream processing of Fc-part containing biopharmaceuticals (mAbs and fusion proteins), F was used as affinity resin. The schematic work-flow is illustrated in Figure 1. Deglycosylation with PNGaseF was performed “on-column”. After washing of bound mAb, N- glycans were released by incubation of the Protein A-mAb complex with PNGaseF at 37 °C followed by elution with H2O, which resulted in higher glycan yields than mAb elution followed

by PNGaseF digestion in solution. PNGaseF was subsequently removed by ultrafiltration. Glycans were dried by vacuum centrifugation and 2-AA labeling was performed via reductive amination by use of the non-toxic reductive agent picoline borane.(31) Excess label was removed by small scale gel filtration, which was performed in custom-made 96-well plates with Sephadex G-10 resin. The last step is a downscaled procedure based on a previously published purification approach.(17, 27) This purification is highly efficient because it separates labeled N-glycans from excess 2-AA in a single centrifugation step.

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Figure 1: Schematic work-flow. Up to 2x 96 samples can be handled simultaneously. Immobilized Protein A or Protein G is used to capture mAbs, Fc-containing fusion proteins or other IgGs with high specificity. Immobilized target is then highly efficiently deglycosylated with the use of PNGaseF. Released N-Glyans are labeled with 2-AA via reductive amination after ultrafiltration to remove remaining proteins. Labeled and purified 2-AA glycans are identified and quantified by RP nanoLC-MS by use of an ion-trap mass spectrometer.